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Layered gadolinium-based nanoparticle as a novel delivery platform for microRNA therapeutics
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 425102 (http://iopscience.iop.org/0957-4484/25/42/425102) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 138.100.4.44 This content was downloaded on 27/12/2014 at 09:46
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 25 (2014) 425102 (12pp)
doi:10.1088/0957-4484/25/42/425102
Layered gadolinium-based nanoparticle as a novel delivery platform for microRNA therapeutics Shannon S Yoo1, Rene Razzak2, Eric Bédard3, Linghong Guo1, Andrew R Shaw4, Ronald B Moore5 and Wilson H Roa1 1
Department of Radiation Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada Division of General Surgery, Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada 3 Division of Thoracic Surgery, Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada 4 Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada 5 Division of Urology, Department of Surgery, University of Alberta Hospital, Alberta, Canada 2
E-mail: [email protected] Received 6 June 2014, revised 8 September 2014 Accepted for publication 8 September 2014 Published 3 October 2014 Abstract
Specific expression patterns of microRNA (miRNA) molecules have been linked to cancer initiation, progression, and metastasis. The accumulating evidence for the role of oncogenic or tumor-suppressing miRNAs identified the need for nano-scaled platform that can help deliver nucleotides to modulate miRNAs. Here we report the synthesis of novel layered gadolinium hydroxychloride (LGdH) nanoparticles, a member of the layered double hydroxide (LDH) family, with physiochemical properties suitable for cell uptake and tracing via magnetic resonance (MR) imaging. As a proof of concept, we demonstrate the inhibition of mature miRNA-10b in metastatic breast cancer cell line using LGdH nanoparticle as a delivery platform. Through characterization analysis, we show that nanoparticles are easily and stably loaded with anti-miRNA oligonucleotides (AMO) and efficiently penetrate cell membranes. We demonstrate that AMOs delivered by LGdH nanoparticles remain functional by inducing changes in the expression of its downstream effector and by curbing the invasive properties. Furthermore, we demonstrate the traceability of LGdH nanoparticles via T1 weighted MR imaging. LGdH nanoparticles, which are biocompatible with cells in vitro, provide a promising multifunctional platform for microRNA therapeutics through their diagnostic, imaging, and therapeutic potentials. Keywords: layered double hydroxide, nanoparticles, anti-microRNA oligonucleotides, breast cancer, T1-MRI, invasion Introduction
miRNAs display increased expression in cancer and act as oncogenes [6, 7]. Studies involving oncogenic miRNAs have demonstrated that anti-miRNA oligonucleotides (AMO) against the mature miRNA sequences can be used to effectively silence miRNAs in vivo [8–10]. Amongst many of the oncogenic miRNAs that have been identified, overexpression of miRNA-10b was shown to specifically promote early stage metastasis in breast cancer [9, 11, 12]. The use of AMOs against miR-10b in these studies was successful in curbing
MicroRNAs (miRNAs) are a class of small, non-coding RNAs that can regulate genes post-transcriptionally by mRNA cleavage or translational repression [1]. Recently, specific expression patterns or mutations of miRNAs were validated as causative factors in a wide variety of cancer malignancies [2, 3] including breast cancer [4, 5]. Although a majority miRNAs act as tumor suppressors, a subset of 0957-4484/14/425102+12$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Nanotechnology 25 (2014) 425102
S S Yoo et al
metastasis to distant organs, which rationalizes the effort to render a suitable delivery vehicle for AMO’s clinical use in order to avoid possible immune response associated with naked oligonucleotides [13]. In this paper, we regulate the miR-10b expression in breast cancer cell lines using a novel delivery vehicle, layered gadolinium hydroxychloride (LGdH) (Gd2(OH)5(H2O)x)Cl nanoparticles. LGdH nanoparticles are structurally similar to a family of layered double hydroxides (LDHs) [14]. LGdH nanoparticle structure consists of positively-charged hydroxide layers of trivalent rare-earth gadolinium ions (Gd3+) with water molecules and exchangeable chloride ions in the interlayer space [15]. The hydrated anions in the interlayer space of LDHs are highly mobile, which allows for incorporation of desired anionic species by a direct ion exchange method of intercalation [16, 17]. The kinetics and mechanisms of ion exchange reaction between LDHs and various kinds of organic and inorganic anions have been extensively studied [16, 18–20]. The favorable electrostatic interactions between multivalent anionic biomolecules and cationic metal hydroxide layers owing to an ion exchange reaction has shown to increase stabilization energy [17] and lower thermodynamic energy [21]. As a result of this desirable property, LDHs need not be conjugated to gene therapy agents in order to deliver them. Many studies have demonstrated success in exploiting the strong anion exchange capability of LDHs by using it as a carrier of a range of gene therapy agents such as antisense oligonucleotide [22], plasmid DNA [23], and siRNA [24]. The family of LDH nanoparticles have several advantages over other vector formulations for the purposes of gene therapy. As a non-viral vector, LDH nanoparticles can minimize the side effects associated with immune response to the virus [25]. With the demonstrated biocompatibility and minimal cell toxicity [14], LDHs nanoparticles has been successfully used in a wide range of therapeutic applications [26, 27]. The shape of LDH nanoparticle can be controlled during the synthesis to target a specific sub-cellular compartment [28], which allows for delivery in a manner specific to the function of the loaded agent. The positive charge of the LDH nanoparticle surface is complementary with the negatively-charged cellular membrane [29], which encourage cellular uptake through clathrin-mediated endocytosis [30]. Furthermore, LDHs can effectively protect the agents from degradation during the uptake process [31, 32], whilst allowing for their recovery at the desired location [17] upon dissolution of LDHs in a low pH environment [14]. The intrinsic properties of LDHs make them suitable for cellular delivery applications. In this paper we present the synthesis of a unique Gd3+based LGdH nanoparticle and characterization of LGdH nanoparticles loaded with AMOs against miR-10b. As a material of high atomic number, rare-earth Gd3+-based nanoparticles are of great interest for their magnetic resonance (MR) imaging contrast [14, 33] and radiation-therapy enhacing capabilities [34, 35]. In this paper, we explore the application of LGdH nanoparticles by examining the delivery efficiencies and testing the functionality of LGdH
nanoparticles loaded with AMO in vitro, and validating its clinical applicability via MR imaging.
Material and methods Preparation and characterization of LGdH and AMO-loaded LGdH nanoparticles
LGdH nanoparticles were synthesized using a previously reported procedure with minor modifications [14]. Briefly, 5.75 ml of 1.5 M NaOH was added to 20 ml of 0.125 M GdCl3·6H2O with vigorous stirring at room temperature to adjust the final pH to 6.5. The precipitate was heated at 80 °C for 2 h with vigorous stirring. The slurry of LGdH was centrifuged at 6000 rpm for 10 min and the supernatant was decanted. Nanoparticle pellet was washed thoroughly with deionized water and dispersed with a sonic dismembrator (Model 705, Fisher Scientific). The sizes of LGdH nanoparticles were determined by using the ellipse tool in the ImageJ program (Ver 1.48d, National Institute of Health). A single-stranded miR-10b inhibitor, miRCURY LNA miRNA Inhibitor with the sequence of 5’ACAAATTCGGTTCTACAGGGT-3’ was used as AMO (Exiqon, Woburn, MA, USA). The equivalent oligodeoxynucleotidebased sequences in untagged and 5’ Alexa Fluor 488-modified forms were used for characterization, confocal microscopy, and flow cytometry studies (Integrated DNA technologies). AMOs were loaded to LGdH nanoparticles via ion exchange. AMO and nanoparticle were mixed in deionized water (pH 7.0) at 1:1 mass ratio and vortexed immediately at the highest setting. The mixture was then shaken on a roller for 30 min, centrifuged, and resuspended in saline at room temperature prior to use. The powdered LGdH and AMO-LGdH nanoparticle samples for x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were obtained by vacuum drying the sample at room temperature. The XRD analysis was conducted on a Rigaku Ultima IV Powder XRD system run at 38 kV and 38 mA using a x-ray generator with a cobalt tube. The search and match was done with JADE software (version 6.1.7601, Materials Data). The FTIR spectrophotometry data (Nicolet 8700, Thermo Scientific) was analyzed via OMNIC software (version 6.2, Thermo Electron Corporation). Zeta potential measurements were recorded at 25 °C in triplicates (Zetasizer Nano-ZS, Malvern Instruments). The average of zeta potential measurements is reported ± standard error of mean. AMO loading efficiency
For the determination of AMO loading and entrapment efficiencies, amount of AMO in the supernatant was assessed via Beckman UV spectrophotometer. Calculations were done according to the following [36]: Loading efficiency(%) Mass AMO added − Mass AMO in supernatant = Mass LGdH nanoparticles × 100. 2
Nanotechnology 25 (2014) 425102
S S Yoo et al
LGdH concentration of 9 μg mL−1. Transfections using Lipofectamine RNAiMAX (Life Technologies) were performed following the manufacturer’s protocol. About onehalf of AMO in AMO-LGdH nanoparticles was used for AMO-lipofectamine and AMO-only groups to compensate for the loading efficiencies. Cell transfections were complete in 24 h.
Entrapment efficiency(%) Mass AMO added − Mass AMO in supernatant = Mass AMO added × 100.
Experiment was conducted in triplicates. Cell culture
Migration and invasion assay
Human breast cancer cell lines MDA-MB-231 and MCF7 were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 1% antibiotic-antimycotic (Invitrogen), 5 μg ml−1 plasmocin prophylactic (Invivogen), and 10% fetal bovine serum (FBS; Gibco). Cells were kept in polystyrene flasks (Corning) in incubator with 5% CO2 at 37 °C.
Cells were starved for 24 h in serum-free DMEM medium prior to the assay. Both migration (8 μm Mililcell cell culture inserts, Merck Millipore) and invasion assays (8 μm QCM™ High Sensitivity Non-cross-linked Collagen Invasion Assay, Merck Millipore) were conducted as per manufacturer’s instructions. Briefly, after plating cells in inserts, cells were incubated for 6 h for the migration assay and 72 h for an invasion assay. Media supplemented with 10% FBS and 20% FBS was used as a chemo-attractant for the migration and invasion assays, respectively. For the migration assay, inserts were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature, permeabilized with 100% methanol, and stained with crystal violet. For the invasion assay, staining reagent in the kit was used as per manufacturer’s instructions. Cotton swabs were used to scrape off nonmigrated cells from the top membrane prior to imaging under Olympus IX70 microscope. One center field views was taken per well using Olympus Plan 10X/0.25PhI or UPlanFl 20x/ 0.5 lenses, Zeiss Axiocam MRc camera, and AxioVision software (Version 4.4.0.0, Carl Zeiss). Both assays were conducted in triplicates. The numbers of migrated cells were counted with Metamorph software (64-bit Version 7.8.4.0; Molecular Devices).
Cell uptake
For preparation for TEM, MDA-MB-231 cells were seeded onto sterile plastic cover slips in a six-well plate 24 h before the treatment. On the day of the treatment, cells were treated with 200 μg ml−1 LGdH nanoparticle and incubated for 1, 6, 24, or 48 h. The cells on cover slips were washed twice with PBS, and then fixed with 2% paraformaldehyde and 2.5% glutaraldehyde. The cells were then dehydrated with an ethanol and block stained with 1% uranyl acetate, infiltrated by Spurrs’ resin, and polymerized in 65°–70 °C oven. Thin sections of 100 nm were cut with ultramicrotome (Leica EM UC6) and coated on a 400 mesh copper grid. The sections were stained with 2% Uranyl acetate and 1% lead citrate. Transmission electron microscopy
The images were taken using JEM 2100 transmission electron microscope at 120 kV (JEOL).
Confocal imaging
MDA-MB-231 cells were seeded onto glass cover slips (Corning) 24 h prior to treatment in a six-well plate. The day after transfection, the media was removed and the wells were washed three times with warm phosphate buffered saline (PBS; Gibco, USA). Cells were fixed in warm 4% paraformaldehyde (Sigma Aldrich) in PBS for 20 min The cover slips were washed twice in PBS, and then mounted on glass slides using a mounting medium with DAPI (Life Technologies). Images were acquired on a Zeiss LSM-710 laser scanning microscope (Carl Zeiss) with 40 × 1.3 Oil DIC M27 using ZEN 2011 software (black edition 64-bit, version 7.0.4.0, Carl Zeiss). All treatment groups were prepared in triplicates.
In vitro cell toxicity
Viability of cells treated with 20, 50, 100, or 200 μg ml−1 concentrations of LGdH nanoparticles or AMO-loaded LGdH nanoparticles was determined using the AlamarBlue Cell Viability Reagent (Invitrogen) as per manufacturer’s instructions. Briefly, MDA-MB-231 cells were seeded at 4 × 103 cells per well on a 96-well assay plate (Corning) and allowed to attach for 24 h. On the next day, cells were treated and incubated for another 48 h. After the addition of AlamarBlue reagent, cells were incubated for 4 h. Absorbance was monitored at 590 nm using Optima 96-well plate reader (BMG LABTECH). Experiment was conducted in quadruplicates.
Flow cytometry Cell transfections
Flow Cytometry was conducted to determine the cell uptake of 5’ Alexa Fluor 488-modified AMO-LGdH nanoparticles. The day after transfection, cells were trypsinized and centrifuged at 200 g for 5 mins. After removal of the supernatant, cells were washed three times with PBS. Cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) with a 15 mW air-cool argon-ion laser at an
MDA-MB-231 cells were seeded onto six-well culture plates (105 cells per well) and allowed to attach for 24 h. Media in all the wells were replaced with DMEM supplemented with 10% FBS without antibiotics prior to transfections. AMOLGdH nanoparticle complexes were prepared at 18 μg: 18 μg weight ratios, and were added to each well to give a final 3
Nanotechnology 25 (2014) 425102
S S Yoo et al
Figure 1. TEM images of (A) pristine LGdH nanoparticles and (B) the same LGdH nanoparticles associated with AMO. The morphology of
LGdH nanoparticles remains the same upon association with AMOs. (C) AMO loading and entrapment percentages for a range of AMO to LGdH nanoparticle mass ratio preparations.
Results
excitation frequency of 488 nm. Fluorescence signals were detected using a band-pass filter 530/30 nm. The BD FACSCalibur™ flow cytometer was automatically set up and compensated with BD Calibrite™ beads used with FACSComp™ software (BD Biosciences). Experiments were conducted in triplicate.
Characterization of AMO-loaded LGdH nanoparticles
The protocol described in Materials and Methods produced homogenous and stable aqueous suspension of oval LGdH nanoparticles (figure 1(A)). The LGdH nanoparticles had an average major and minor diameters of 146 nm and 90 nm, respectively, within the range for effective endocytosis [29]. We tested several AMO to LGdH nanoparticle mass ratios to assess the loading and entrapment efficiencies. The loading efficiency was highest for the highest AMO to LGdH nanoparticle ratio tested (6:1), whereas the entrapment efficiency was highest for the lowest AMO to LGdH nanoparticle ratio tested (0.5:1) (figure 1(C)). At 1:1 AMO to LGdH nanoparticle mass ratio, the loading and entrapment efficiencies were 41.8% and 89.5%, respectively, and this ratio was selected for all the subsequent experiments. The apparent size and shape of LGdH nanoparticles did not change after the addition of AMO (AMO-LGdH; figure 1(B)), which indicated effective intercalation of AMOs into the interlayer spaces of LGdH nanoparticles [24]. Intercalation activity of AMOs into LGdH nanoparticles was further confirmed by the absence of agglomeration, which has reported to occur for LDHs subjected to suboptimal intercalation activity [37]. In line with previous observations for LDHs loaded with anionic biomolecules [17, 22, 24], a decrease in positive surface charge was observed for our AMO-loaded LGdH nanoparticles. The LGdH nanoparticles loaded with AMOs as measured by zeta potential had more neutral electrical characteristic in an aqueous solution with zeta potentials of +14.6 ± 0.1 mV compared to +45.6 ± 0.4 mV for pristine LGdH nanoparticles (figure 2). Further characterization was conducted to confirm the association of AMOs with LGdH nanoparticles. The FTIR spectrum of the both samples showed absorption peaks that are characteristic of rare earth metal-based hydroxycarbonate nanoparticles with the vibration of the OH-group at 3525 cm−1 and 3497 cm−1 (figure 3) [38]. From the analysis of deconvolved FTIR spectra of the AMO-loaded LGdH nanoparticles, we were able to identify peaks that were absent in pure LGdH spectra that could specifically be attributed to nucleic acids (data not displayed). The bands at 1052 cm−1
Western blotting
Cells were washed twice with PBS (Gibco) and whole cell lysates were harvested in lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 nM Tris, pH 8.0) using a cell scraper (Corning). Protease inhibitor cocktail (Sigma Aldrich) was added to the collection tube. Proteins were resolved by Mini-Protean TGX 4–20% gel (Biorad), transferred to PVDF membrane (Biorad), blocked in 5% skim milk in TBST, and blotted with antibodies for HOXD10 (1:1500, Abcam) and αtubulin (1:2000, Abcam), and secondary antibody conjugated to HRP (1:2000, Abcam). Clarity Western ECL Substrate (Biorad) was used for film-based imaging. MR imaging
Magnetic strength measurements were made with vibrating sample magnetometer (VSM 7400, Lakeshore Cryotronics) at room temperature. 3.0T Philips MRI using spin-echo sequence was used to acquire T1-weighted image (TE, 8 ms; TR, 800 ms). Statistical analysis
Statistical significance for migration and invasion were determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test with Graphpad Prism software (Prism 5 for Windows, Version 5.04, GraphPad Software). Statistical significance for cell toxicity was determined by a two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison tests. P values of
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My IOPscience
Layered gadolinium-based nanoparticle as a novel delivery platform for microRNA therapeutics
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 Nanotechnology 25 425102 (http://iopscience.iop.org/0957-4484/25/42/425102) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 138.100.4.44 This content was downloaded on 27/12/2014 at 09:46
Please note that terms and conditions apply.
Nanotechnology Nanotechnology 25 (2014) 425102 (12pp)
doi:10.1088/0957-4484/25/42/425102
Layered gadolinium-based nanoparticle as a novel delivery platform for microRNA therapeutics Shannon S Yoo1, Rene Razzak2, Eric Bédard3, Linghong Guo1, Andrew R Shaw4, Ronald B Moore5 and Wilson H Roa1 1
Department of Radiation Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada Division of General Surgery, Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada 3 Division of Thoracic Surgery, Department of Surgery, University of Alberta Hospital, Edmonton, Alberta, Canada 4 Department of Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada 5 Division of Urology, Department of Surgery, University of Alberta Hospital, Alberta, Canada 2
E-mail: [email protected] Received 6 June 2014, revised 8 September 2014 Accepted for publication 8 September 2014 Published 3 October 2014 Abstract
Specific expression patterns of microRNA (miRNA) molecules have been linked to cancer initiation, progression, and metastasis. The accumulating evidence for the role of oncogenic or tumor-suppressing miRNAs identified the need for nano-scaled platform that can help deliver nucleotides to modulate miRNAs. Here we report the synthesis of novel layered gadolinium hydroxychloride (LGdH) nanoparticles, a member of the layered double hydroxide (LDH) family, with physiochemical properties suitable for cell uptake and tracing via magnetic resonance (MR) imaging. As a proof of concept, we demonstrate the inhibition of mature miRNA-10b in metastatic breast cancer cell line using LGdH nanoparticle as a delivery platform. Through characterization analysis, we show that nanoparticles are easily and stably loaded with anti-miRNA oligonucleotides (AMO) and efficiently penetrate cell membranes. We demonstrate that AMOs delivered by LGdH nanoparticles remain functional by inducing changes in the expression of its downstream effector and by curbing the invasive properties. Furthermore, we demonstrate the traceability of LGdH nanoparticles via T1 weighted MR imaging. LGdH nanoparticles, which are biocompatible with cells in vitro, provide a promising multifunctional platform for microRNA therapeutics through their diagnostic, imaging, and therapeutic potentials. Keywords: layered double hydroxide, nanoparticles, anti-microRNA oligonucleotides, breast cancer, T1-MRI, invasion Introduction
miRNAs display increased expression in cancer and act as oncogenes [6, 7]. Studies involving oncogenic miRNAs have demonstrated that anti-miRNA oligonucleotides (AMO) against the mature miRNA sequences can be used to effectively silence miRNAs in vivo [8–10]. Amongst many of the oncogenic miRNAs that have been identified, overexpression of miRNA-10b was shown to specifically promote early stage metastasis in breast cancer [9, 11, 12]. The use of AMOs against miR-10b in these studies was successful in curbing
MicroRNAs (miRNAs) are a class of small, non-coding RNAs that can regulate genes post-transcriptionally by mRNA cleavage or translational repression [1]. Recently, specific expression patterns or mutations of miRNAs were validated as causative factors in a wide variety of cancer malignancies [2, 3] including breast cancer [4, 5]. Although a majority miRNAs act as tumor suppressors, a subset of 0957-4484/14/425102+12$33.00
1
© 2014 IOP Publishing Ltd Printed in the UK
Nanotechnology 25 (2014) 425102
S S Yoo et al
metastasis to distant organs, which rationalizes the effort to render a suitable delivery vehicle for AMO’s clinical use in order to avoid possible immune response associated with naked oligonucleotides [13]. In this paper, we regulate the miR-10b expression in breast cancer cell lines using a novel delivery vehicle, layered gadolinium hydroxychloride (LGdH) (Gd2(OH)5(H2O)x)Cl nanoparticles. LGdH nanoparticles are structurally similar to a family of layered double hydroxides (LDHs) [14]. LGdH nanoparticle structure consists of positively-charged hydroxide layers of trivalent rare-earth gadolinium ions (Gd3+) with water molecules and exchangeable chloride ions in the interlayer space [15]. The hydrated anions in the interlayer space of LDHs are highly mobile, which allows for incorporation of desired anionic species by a direct ion exchange method of intercalation [16, 17]. The kinetics and mechanisms of ion exchange reaction between LDHs and various kinds of organic and inorganic anions have been extensively studied [16, 18–20]. The favorable electrostatic interactions between multivalent anionic biomolecules and cationic metal hydroxide layers owing to an ion exchange reaction has shown to increase stabilization energy [17] and lower thermodynamic energy [21]. As a result of this desirable property, LDHs need not be conjugated to gene therapy agents in order to deliver them. Many studies have demonstrated success in exploiting the strong anion exchange capability of LDHs by using it as a carrier of a range of gene therapy agents such as antisense oligonucleotide [22], plasmid DNA [23], and siRNA [24]. The family of LDH nanoparticles have several advantages over other vector formulations for the purposes of gene therapy. As a non-viral vector, LDH nanoparticles can minimize the side effects associated with immune response to the virus [25]. With the demonstrated biocompatibility and minimal cell toxicity [14], LDHs nanoparticles has been successfully used in a wide range of therapeutic applications [26, 27]. The shape of LDH nanoparticle can be controlled during the synthesis to target a specific sub-cellular compartment [28], which allows for delivery in a manner specific to the function of the loaded agent. The positive charge of the LDH nanoparticle surface is complementary with the negatively-charged cellular membrane [29], which encourage cellular uptake through clathrin-mediated endocytosis [30]. Furthermore, LDHs can effectively protect the agents from degradation during the uptake process [31, 32], whilst allowing for their recovery at the desired location [17] upon dissolution of LDHs in a low pH environment [14]. The intrinsic properties of LDHs make them suitable for cellular delivery applications. In this paper we present the synthesis of a unique Gd3+based LGdH nanoparticle and characterization of LGdH nanoparticles loaded with AMOs against miR-10b. As a material of high atomic number, rare-earth Gd3+-based nanoparticles are of great interest for their magnetic resonance (MR) imaging contrast [14, 33] and radiation-therapy enhacing capabilities [34, 35]. In this paper, we explore the application of LGdH nanoparticles by examining the delivery efficiencies and testing the functionality of LGdH
nanoparticles loaded with AMO in vitro, and validating its clinical applicability via MR imaging.
Material and methods Preparation and characterization of LGdH and AMO-loaded LGdH nanoparticles
LGdH nanoparticles were synthesized using a previously reported procedure with minor modifications [14]. Briefly, 5.75 ml of 1.5 M NaOH was added to 20 ml of 0.125 M GdCl3·6H2O with vigorous stirring at room temperature to adjust the final pH to 6.5. The precipitate was heated at 80 °C for 2 h with vigorous stirring. The slurry of LGdH was centrifuged at 6000 rpm for 10 min and the supernatant was decanted. Nanoparticle pellet was washed thoroughly with deionized water and dispersed with a sonic dismembrator (Model 705, Fisher Scientific). The sizes of LGdH nanoparticles were determined by using the ellipse tool in the ImageJ program (Ver 1.48d, National Institute of Health). A single-stranded miR-10b inhibitor, miRCURY LNA miRNA Inhibitor with the sequence of 5’ACAAATTCGGTTCTACAGGGT-3’ was used as AMO (Exiqon, Woburn, MA, USA). The equivalent oligodeoxynucleotidebased sequences in untagged and 5’ Alexa Fluor 488-modified forms were used for characterization, confocal microscopy, and flow cytometry studies (Integrated DNA technologies). AMOs were loaded to LGdH nanoparticles via ion exchange. AMO and nanoparticle were mixed in deionized water (pH 7.0) at 1:1 mass ratio and vortexed immediately at the highest setting. The mixture was then shaken on a roller for 30 min, centrifuged, and resuspended in saline at room temperature prior to use. The powdered LGdH and AMO-LGdH nanoparticle samples for x-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were obtained by vacuum drying the sample at room temperature. The XRD analysis was conducted on a Rigaku Ultima IV Powder XRD system run at 38 kV and 38 mA using a x-ray generator with a cobalt tube. The search and match was done with JADE software (version 6.1.7601, Materials Data). The FTIR spectrophotometry data (Nicolet 8700, Thermo Scientific) was analyzed via OMNIC software (version 6.2, Thermo Electron Corporation). Zeta potential measurements were recorded at 25 °C in triplicates (Zetasizer Nano-ZS, Malvern Instruments). The average of zeta potential measurements is reported ± standard error of mean. AMO loading efficiency
For the determination of AMO loading and entrapment efficiencies, amount of AMO in the supernatant was assessed via Beckman UV spectrophotometer. Calculations were done according to the following [36]: Loading efficiency(%) Mass AMO added − Mass AMO in supernatant = Mass LGdH nanoparticles × 100. 2
Nanotechnology 25 (2014) 425102
S S Yoo et al
LGdH concentration of 9 μg mL−1. Transfections using Lipofectamine RNAiMAX (Life Technologies) were performed following the manufacturer’s protocol. About onehalf of AMO in AMO-LGdH nanoparticles was used for AMO-lipofectamine and AMO-only groups to compensate for the loading efficiencies. Cell transfections were complete in 24 h.
Entrapment efficiency(%) Mass AMO added − Mass AMO in supernatant = Mass AMO added × 100.
Experiment was conducted in triplicates. Cell culture
Migration and invasion assay
Human breast cancer cell lines MDA-MB-231 and MCF7 were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 1% antibiotic-antimycotic (Invitrogen), 5 μg ml−1 plasmocin prophylactic (Invivogen), and 10% fetal bovine serum (FBS; Gibco). Cells were kept in polystyrene flasks (Corning) in incubator with 5% CO2 at 37 °C.
Cells were starved for 24 h in serum-free DMEM medium prior to the assay. Both migration (8 μm Mililcell cell culture inserts, Merck Millipore) and invasion assays (8 μm QCM™ High Sensitivity Non-cross-linked Collagen Invasion Assay, Merck Millipore) were conducted as per manufacturer’s instructions. Briefly, after plating cells in inserts, cells were incubated for 6 h for the migration assay and 72 h for an invasion assay. Media supplemented with 10% FBS and 20% FBS was used as a chemo-attractant for the migration and invasion assays, respectively. For the migration assay, inserts were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature, permeabilized with 100% methanol, and stained with crystal violet. For the invasion assay, staining reagent in the kit was used as per manufacturer’s instructions. Cotton swabs were used to scrape off nonmigrated cells from the top membrane prior to imaging under Olympus IX70 microscope. One center field views was taken per well using Olympus Plan 10X/0.25PhI or UPlanFl 20x/ 0.5 lenses, Zeiss Axiocam MRc camera, and AxioVision software (Version 4.4.0.0, Carl Zeiss). Both assays were conducted in triplicates. The numbers of migrated cells were counted with Metamorph software (64-bit Version 7.8.4.0; Molecular Devices).
Cell uptake
For preparation for TEM, MDA-MB-231 cells were seeded onto sterile plastic cover slips in a six-well plate 24 h before the treatment. On the day of the treatment, cells were treated with 200 μg ml−1 LGdH nanoparticle and incubated for 1, 6, 24, or 48 h. The cells on cover slips were washed twice with PBS, and then fixed with 2% paraformaldehyde and 2.5% glutaraldehyde. The cells were then dehydrated with an ethanol and block stained with 1% uranyl acetate, infiltrated by Spurrs’ resin, and polymerized in 65°–70 °C oven. Thin sections of 100 nm were cut with ultramicrotome (Leica EM UC6) and coated on a 400 mesh copper grid. The sections were stained with 2% Uranyl acetate and 1% lead citrate. Transmission electron microscopy
The images were taken using JEM 2100 transmission electron microscope at 120 kV (JEOL).
Confocal imaging
MDA-MB-231 cells were seeded onto glass cover slips (Corning) 24 h prior to treatment in a six-well plate. The day after transfection, the media was removed and the wells were washed three times with warm phosphate buffered saline (PBS; Gibco, USA). Cells were fixed in warm 4% paraformaldehyde (Sigma Aldrich) in PBS for 20 min The cover slips were washed twice in PBS, and then mounted on glass slides using a mounting medium with DAPI (Life Technologies). Images were acquired on a Zeiss LSM-710 laser scanning microscope (Carl Zeiss) with 40 × 1.3 Oil DIC M27 using ZEN 2011 software (black edition 64-bit, version 7.0.4.0, Carl Zeiss). All treatment groups were prepared in triplicates.
In vitro cell toxicity
Viability of cells treated with 20, 50, 100, or 200 μg ml−1 concentrations of LGdH nanoparticles or AMO-loaded LGdH nanoparticles was determined using the AlamarBlue Cell Viability Reagent (Invitrogen) as per manufacturer’s instructions. Briefly, MDA-MB-231 cells were seeded at 4 × 103 cells per well on a 96-well assay plate (Corning) and allowed to attach for 24 h. On the next day, cells were treated and incubated for another 48 h. After the addition of AlamarBlue reagent, cells were incubated for 4 h. Absorbance was monitored at 590 nm using Optima 96-well plate reader (BMG LABTECH). Experiment was conducted in quadruplicates.
Flow cytometry Cell transfections
Flow Cytometry was conducted to determine the cell uptake of 5’ Alexa Fluor 488-modified AMO-LGdH nanoparticles. The day after transfection, cells were trypsinized and centrifuged at 200 g for 5 mins. After removal of the supernatant, cells were washed three times with PBS. Cells were analyzed by a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) with a 15 mW air-cool argon-ion laser at an
MDA-MB-231 cells were seeded onto six-well culture plates (105 cells per well) and allowed to attach for 24 h. Media in all the wells were replaced with DMEM supplemented with 10% FBS without antibiotics prior to transfections. AMOLGdH nanoparticle complexes were prepared at 18 μg: 18 μg weight ratios, and were added to each well to give a final 3
Nanotechnology 25 (2014) 425102
S S Yoo et al
Figure 1. TEM images of (A) pristine LGdH nanoparticles and (B) the same LGdH nanoparticles associated with AMO. The morphology of
LGdH nanoparticles remains the same upon association with AMOs. (C) AMO loading and entrapment percentages for a range of AMO to LGdH nanoparticle mass ratio preparations.
Results
excitation frequency of 488 nm. Fluorescence signals were detected using a band-pass filter 530/30 nm. The BD FACSCalibur™ flow cytometer was automatically set up and compensated with BD Calibrite™ beads used with FACSComp™ software (BD Biosciences). Experiments were conducted in triplicate.
Characterization of AMO-loaded LGdH nanoparticles
The protocol described in Materials and Methods produced homogenous and stable aqueous suspension of oval LGdH nanoparticles (figure 1(A)). The LGdH nanoparticles had an average major and minor diameters of 146 nm and 90 nm, respectively, within the range for effective endocytosis [29]. We tested several AMO to LGdH nanoparticle mass ratios to assess the loading and entrapment efficiencies. The loading efficiency was highest for the highest AMO to LGdH nanoparticle ratio tested (6:1), whereas the entrapment efficiency was highest for the lowest AMO to LGdH nanoparticle ratio tested (0.5:1) (figure 1(C)). At 1:1 AMO to LGdH nanoparticle mass ratio, the loading and entrapment efficiencies were 41.8% and 89.5%, respectively, and this ratio was selected for all the subsequent experiments. The apparent size and shape of LGdH nanoparticles did not change after the addition of AMO (AMO-LGdH; figure 1(B)), which indicated effective intercalation of AMOs into the interlayer spaces of LGdH nanoparticles [24]. Intercalation activity of AMOs into LGdH nanoparticles was further confirmed by the absence of agglomeration, which has reported to occur for LDHs subjected to suboptimal intercalation activity [37]. In line with previous observations for LDHs loaded with anionic biomolecules [17, 22, 24], a decrease in positive surface charge was observed for our AMO-loaded LGdH nanoparticles. The LGdH nanoparticles loaded with AMOs as measured by zeta potential had more neutral electrical characteristic in an aqueous solution with zeta potentials of +14.6 ± 0.1 mV compared to +45.6 ± 0.4 mV for pristine LGdH nanoparticles (figure 2). Further characterization was conducted to confirm the association of AMOs with LGdH nanoparticles. The FTIR spectrum of the both samples showed absorption peaks that are characteristic of rare earth metal-based hydroxycarbonate nanoparticles with the vibration of the OH-group at 3525 cm−1 and 3497 cm−1 (figure 3) [38]. From the analysis of deconvolved FTIR spectra of the AMO-loaded LGdH nanoparticles, we were able to identify peaks that were absent in pure LGdH spectra that could specifically be attributed to nucleic acids (data not displayed). The bands at 1052 cm−1
Western blotting
Cells were washed twice with PBS (Gibco) and whole cell lysates were harvested in lysis buffer (150 mM sodium chloride, 1% Triton X-100, 50 nM Tris, pH 8.0) using a cell scraper (Corning). Protease inhibitor cocktail (Sigma Aldrich) was added to the collection tube. Proteins were resolved by Mini-Protean TGX 4–20% gel (Biorad), transferred to PVDF membrane (Biorad), blocked in 5% skim milk in TBST, and blotted with antibodies for HOXD10 (1:1500, Abcam) and αtubulin (1:2000, Abcam), and secondary antibody conjugated to HRP (1:2000, Abcam). Clarity Western ECL Substrate (Biorad) was used for film-based imaging. MR imaging
Magnetic strength measurements were made with vibrating sample magnetometer (VSM 7400, Lakeshore Cryotronics) at room temperature. 3.0T Philips MRI using spin-echo sequence was used to acquire T1-weighted image (TE, 8 ms; TR, 800 ms). Statistical analysis
Statistical significance for migration and invasion were determined by a one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test with Graphpad Prism software (Prism 5 for Windows, Version 5.04, GraphPad Software). Statistical significance for cell toxicity was determined by a two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison tests. P values of
